Recent reports indicating a rise in infections caused by carbapenem- resistant Klebsiella pneumoniae (KPC) in the United States, specifically the New York metropolitan area, and elsewhere have alerted clinicians, infection control teams and public health officials. KPCs typically exhibit a wide spectrum of antimicrobial resistance, and pan-resistance in some cases, thereby severely impacting treatment options and outcomes. A troubling aspect is that the resistance determinant, blaKPC is on a transposon that is harbored on transmissible plasmids. These resistance bearing plasmids have transmitted to other bacterial genera, such as E. coli. Therefore, it is critical to understand the nature and the genetic basis of spread and the acquisition of these resistance-determining elements. The goal of the present proposal is to determine the role of strains, plasmids, and transposons in the spread and emergence of blaKPC among KPC and other genera. These studies will be done in the New York area, the current epicenter of KPC strains in the US and globally. We will characterize the nature and extent of the KPC epidemic in two consecutive cross-sectional studies (years 1 and 4) to examine the molecular epidemiology of infecting isolates over the 5-year project period, including other emerging carbapenem-resistant Enterobacteriaceae. These studies will involve extensive molecular characterization of the isolates, physical and genetic mapping of resistance bearing mobile elements and determinants, including evaluating carbapenemase activity, and determining the relative virulence of circulating strains in a neutrophil model of infection. To determine whether predominant KPC clones or blaKPC-harboring plasmids have unique genomic content or organization we will conduct de novo whole genome sequencing (WGS) of major KPC strains and their plasmids. In addition, in order to examine the evolutionary trajectories (expansion and diversification) of the current epidemic clones we will perform high- throughput comparative WGS of major KPC strains using clone-specific reference strains. The information gained from the molecular epidemiologic studies on KPC strains and carbapenem-resistant Enterobacteriaceae, blaKPC -harboring plasmids, transposons and other resistance determinants will help evaluate the extent to which KPC strains are attributed to primary (clonal) transmission or acquired resistance and whether any new genotypes are emerging in this high incidence region. Our neutrophil studies will determine whether host-pathogen interaction play a role in the current overrepresentation of genotypes. The results from the genomic studies may help elucidate factors that contribute to the dissemination of predominant clones of KPC that are currently fueling the epidemic. Furthermore, deep molecular dissection of major strains will indicate patterns of genetic diversification and demonstrate clonal emergence. Accomplishing these aims will deepen our understanding of a critical, public health problem by detailing the molecular and genetic characteristics of KPC isolates in the epicenter of a developing crisis; assessing the relative virulence of circulating strains; and by examining the lateral transfer of blaKPC genes to other members of Enterobacteriaceae, events that would significantly hinder treatment and infection control programs. These data can inform current and future control strategies and in the development of rapid diagnostics.